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Single molecule biophysics

Assembly of endocytic machinery around individual influenza viruses during viral entry. Single molecule biophysics. Influenza Virus – www.medizin.de. Influenza virus life cycle. www.northwestern.edu. Aim of study. Aims:

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Single molecule biophysics

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  1. Assembly of endocytic machinery around individual influenza viruses during viral entry Single molecule biophysics Influenza Virus – www.medizin.de

  2. Influenza viruslife cycle www.northwestern.edu

  3. Aim of study • Aims: • To characterize endocytic mechanism of Influenza virus infection - on a single-virus level • To elucidate through which pathways endocytosis occurs • Clathrin-mediated • Clathrin- and caveolin-independent • Formation of clathrin coated pits Dr. Helge Gad, Karolinska Institute, Stockholm

  4. CCP / CCV flourescence Test of EYFP incorporation into clathrin-coated pits A: Immunoflourescence (Ab) of clathrin-coated pits B: EYFP-clathrin, transfected cell C: overlay A/B (yellow) D: Transferrin (red) internalization in EYFP-clathrin (green) cell

  5. Visualizing influenza • Virus labelling • DiD (lipophilic)

  6. 65%: clathrin-associated 35%: no association 3-stage process I: actin-dependent surface movement II: rapid unidirectional, towards perinuclear region III: bidirectional microtubule-dependent Entry pathways

  7. Formationof CCPs • Formation of CCPs: • 94% de novo formation of CCPs • Prevalence at random sites ~1:20 • Prevalence at earlier binding sites ~1:20 • Formation at mobile hospots should cause exp decay

  8. Dynamics of CCPs • Dynamics of CCPs • On average • Initiates 190s after virus binding • Persists for 70s • CCV uncoating in few seconds • Proceed to stage II within 40s • Longer time before stage II could be caused by recycling and a second endocytosis

  9. Neuraminidase effect • Neuraminidase effect • Cleave multivalent bands between HA and cell surface sialic acid • Thereby facilitating viral movement? • NA inhibitor  no effect on endocytosis

  10. Test for viral fusion in both pathways Quenching by high density viral surface DiD 65% clathrin-associated 35% non-associated Viral fusion

  11. Equal binding time Pathways competing for viral entry Equal distance to nucleus Trafficking kinetics • Equal stage II time • Convergence of endocytic trafficking

  12. Conclusions • Influenza viruses enter cells by at least two endocytic pathways • ~2/3 enters by clathrin-mediated endocytosis • Allmost all CCPs are formed de novo • Pathways have equal kinetics • No NA effect on endocytosis • Signal to clathrin? • Curvature of membrane by multivalent HA binding Yoshihiro Kawaoka, University of Wisconsin-Madison

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